This invention relates to the field of microbial fermentation and biopesticide production. In particular, this invention provides a disposable bioreactor for use in culturing microorganisms and other cells.
Various scientific articles are referred to throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
Current agricultural practices make use of pesticide strategies that are predominantly based on chemicals. Public concern about the environmental hazards of chemical pesticides has increased in recent years. While the range of environmentally safe biological control agents has been growing, growers still rely on the chemical alternatives. Biological control agents offer many advantages over conventional chemical pesticides, but they have been slow to gain acceptance because they are more difficult to produce, distribute and apply.
One biological control agent with great potential is the entomopathogenic nematode. While some species of nematode are phytopathogenic, others are not. Among the non-phytopathogenic species are those which are entomopathogenic and proven useful in controlling pathogenic insects on crop plants. These nematodes, from the families Steinernematidae and Heterorhabditidae, are mutualistically associated with bacteria from the genus Xenorhabdus. Together they have an extraordinarily high host range that spans nearly all the insect orders. This wide host range is a major reason why these families of nematodes have great agricultural and economic value (Kaya and Gaugler, 1993, Ann. Rev. Entomol. 38:181-206).
In spite of these advantages, the use of 10 nematodes as a biological control agent has been slow to develop. Key problems have been the development of up-scalable culturing techniques, stable storage conditions, transporting the product without loss, and reliable application formulations. Production costs per hectare of nematode cultures exceed those of most chemicals. The relatively short shelf-life of nematodes of a few weeks as well as their restricted temperature range necessitates special handling and contributes to inconsistent performance. Formulations designed to improve the stability and performance of the product are expensive. Strains of nematodes that are efficacious when freshly produced and applied in a research scale frequently do not perform well on an industrial scale. These problems restrict the use of nematodes to primarily high-value specialty crops, and even for these crops, the expense and expertise needed to successfully culture nematodes for biocontrol is beyond the scope of the typical grower or local producer of biopesticides.
The development of disposable micro-culture technology would permit local production of nematodes and other biological control agents, and would alleviate many of these problems. Local production eliminates both the hazards of distribution and storage. Fresh cultures would also be in their most efficacious form for applications. Finally, inexpensive small-scale production of the biological control agents eliminates the most costly elements the existing commercial production scheme: formulation, storage, transport, waste disposal and starting capital.
Conventional microbial fermentation systems generally are composed of a rigid container (e.g., stainless steel or glass) having a means for aerating and mixing the culture contents (e.g., an air inlet port and an impeller or some other mixing means). Clearly, such equipment is not intended to be disposable, and therefore is not suitable for on-site use in small scale production of microorganisms. For local micro-culture to be a success, a disposable micro-culture container must be developed. Such a container should be inexpensive, disposable and easy to use. It should be made of a material that is sterilizable, strong enough to allow the scale-up of culture size, and manufacturable by existing technology. Finally, the design should be one that is flexible and will allow the culturing of a wide range of cells under both aerobic and anaerobic conditions.
Previous attempts have been made to develop disposable culture containers for microbial fermentation. For instance U.S. Pat. No. 5,565,015 discloses a disposable fermenter comprising a flat-bottom plastic container with a flexible flat tube for introduction of liquid. G.B. Patent No. 1,380,316 discloses a flat-bottom plastic container having a somewhat elongated, narrow neck terminating in a releasably closeable mouth that serves as a port for addition of liquid and for communication with the outside atmosphere. Though these disposable containers may be suitable for culturing certain microorganisms under specific conditions, they are not suitable for culturing a wide range of microorganisms, particularly those requiring good aeration or gas exchange, and long culture times.
The present invention provides an apparatus and system for culturing cells or microbial organisms that is disposable, easy to use, inexpensive and versatile. The invention enables the preparation of microorganisms by people not specifically trained in microbiology or aseptic technique. It enables microorganisms to be grown safely, for a variety of purposes, without the need for specialized facilities such as temperature controlled rooms, laminar flow cabinets and sterilization equipment.
According to one aspect of the invention, a disposable bioreactor for culturing microorganisms or cells is provided. The bioreactor comprises flexible or semi-flexible waterproof sheets, preferably plastic, sealed along their edges to form a container. In one embodiment, the container has a wedge shaped or rounded bottom to facilitate mixing and aeration by eliminating xe2x80x9cdead spacexe2x80x9d in corners, and features a means located at the lowermost extremity of the rounded or wedge shaped bottom for generating gas bubbles for mixing and providing gases to microbial or cellular liquid cultures within the container. In another embodiment, the container has a flat bottom and features a series of gas bubble generators situated at intervals along the bottom. In each of these embodiments, the gas bubbler or series of bubblers is situated such that gas bubbles are generated at the bottom of the container and rise to the top. During their transit from the bottom to top of the container, the bubbles tend to get smaller, which facilitates gas exchange. In preferred embodiments of the invention, a gas diffuser is operably attached to the bubbler, to break larger bubbles into smaller bubbles.
The container comprises at least one inlet port for introducing gases or liquids and at least one exit port for exhausting gases or removing liquids. The gas inlet port is attached to a gas pump by tubing, which preferably contains one or more filters for removing microbial contaminants from the gas stream. The outlet port further comprises an exhaust tube extending therefrom, between about 3 inches and about 5 feet in length. In a preferred embodiment, the exhaust tube is coiled to serve as a condenser for liquids evaporating during the culturing process. The exhaust tube may further comprise a valve to prevent back flushing of liquids to the interior of the container, and a filter for filtering microbial contaminants.
The disposable bioreactor of the invention also may contain an inoculation port for introducing inoculant into the container. It may further comprise a breakable pouch on an interior face of the container, or a separate external chamber connected by tubing to the bioreactor chamber, for delivery of concentrated or dried growth medium, inoculum, or other substances. Other features are described in detail below.
According to another aspect of the invention, a kit is provided for culturing a microorganism, which comprises: (a) a disposable bioreactor as described above; and (b) instructions for using the disposable bioreactor to culture the microorganism. In various embodiments, the kit further comprises one or more of the following: (c) ingredients for preparing, or prepared culture medium for culturing the microorganism; (d) inoculum of the microorganism to be cultured; (e) accessories for attaching the bioreactor container to a local source of water for filling the container; (f) accessories for connecting the bioreactor inlet port to a gas pump; and (g) a gas pump.
In preferred embodiments, the kit is specifically adapted for culturing a microorganism selected from the group consisting of bacteria, cyanobacteria, fungi, algae, protozoans and nematodes. Most preferably, the kit is adapted for culturing entomopathogenic nematodes for use as biopesticides.
Other features and advantages of the present invention will be better understood by reference to the drawings and detailed description that follow.